microRNA-29b mediates fibrotic induction of human xylosyltransferase-I in human dermal fibroblasts via the Sp1 pathway

Abstract

Diminished microRNA-29b levels have recently been revealed to provoke increased expression and accumulation of extracellular matrix molecules, such as collagens in fibrotic remodeling. Subsequently, the aim of this study was to find out whether microRNA-29b might also regulate human xylosyltransferase (XT)-I expression. XT-I has been characterized previously as a fibrosis biomarker catalyzing the key step of proteoglycan biosynthesis. While we demonstrate that XYLT1 is neither a target of microRNA-29b identified in silico nor a direct 3′ untranslated region binding partner of microRNA-29b, transfection of normal human dermal fibroblasts with microRNA-29b inhibitor strongly increased XYLT1 mRNA expression and XT activity. Combined results of the target prediction analysis and additional transfection experiments pointed out that microRNA-29b exerts indirect influence on XT-I by targeting the transcription factor specificity protein 1 (Sp1). We could confirm our hypothesis due to the decrease in XYLT1 promoter activity after Sp1 binding site mutation and the approval of occupancy of these binding sites by Sp1 in vitro. Taken together, a hitherto unidentified pathway of XT-I regulation via microRNA-29b/Sp1 was determined in this study. Our observations will facilitate the understanding of complex molecular fibrotic pathways and provide new opportunities to investigate microRNA-based antifibrotic tools.

Activin A-Mediated Regulation of XT-I in Human Skin Fibroblasts

“…We found that activin A-mediated XYLT1 mRNA expression increase is time-and concentration-dependent, and considerably increased the intra-and extracellular XT enzyme activity in NHDF. The correlation of mRNA expression and enzyme activity increase regarding XT-I has been shown in this and numerous other studies using TGFβ1-treated human dermal and cardiac fibroblasts [26,27,[45][46][47]. Furthermore, we observed, in consistency with previous results in NHDF [25], that the extracellular enzyme activity of untreated cells increased over time due to the protein accumulation in the cell supernatant, while intracellular XT activity remains constant over the test period of 120 h. These findings support the hypothesis that two regulatory mechanisms exist to control enzyme activity in NHDF: One at the transcriptional stage and another operating post-translationally, shedding the Golgi-resident, constitutive active enzyme from the membrane for release to the extracellular space controlling the rate of PG biosynthesis by reducing the cellular enzyme amount [24,29].…”

“…Many TGFβ superfamily ligands are potent mediators of ECM deposition and are highly up-regulated under fibrotic conditions [42]. Until now, studies have described an induction of XYLT1 expression in fibrotic tissue or in cultured primary cells treated with TGFβ1, IL-1β or thrombin [26,27,[43][44][45][46][47], while the effect of other pro-fibrotic cytokines and their underlying signalling pathways remain unknown.…”

Identification of Putative Non-Substrate-Based XT-I Inhibitors by Natural Product Library Screening

“…Furthermore, this XYLT1 transcription regulation seemed to be isoform-specific because the XYLT2 mRNA expression in the TGF-β1-treated cells was not affected. This correlation of XYLT1 mRNA expression level and enzyme activity has been shown in numerous studies using TGF-β1-treated human dermal and cardiac fibroblasts [ 7 , 18 , 29 , 42 ]. These results clearly show that amphotericin B and, to a smaller extent, celastrol are capable of dual XT-I inhibition via direct protein-ligand binding and XYLT1 transcription regulation.…”

“…Only a few studies have evaluated the role of miRNAs in TGF-β1-driven XYLT1 expression regulation so far. Recent studies by our group have revealed an miRNA-145/KLF4- and miRNA-29b/SP1-mediated XT regulatory pathway in human skin fibroblasts [ 42 , 69 ], while Theis et al demonstrated XYLT1 to be a direct target of miRNA-133b in the context of murine spinal cord injury [ 70 ]. In summary, we were not only able to identify putative XT-I inhibitors in this study but also found first evidence of a regulatory mechanism of TGF-β1-induced XYLT1 expression by miRNA-21.…”

“…The RNA extraction from cell lysates and cDNA synthesis for mRNA and miRNA expression analysis were performed as described previously [ 7 , 42 ].…”